Heterologous expression from the human D-Loop in organello

Jackson, Christopher; Zbinden, C; Gallati, Sabina; Schaller, André (2014). Heterologous expression from the human D-Loop in organello. Mitochondrion, 17, pp. 67-75. Elsevier 10.1016/j.mito.2014.05.011

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We report the expression of a linear reporter construct in isolated human mitochondria. The reporter construct contained the entire human D-Loop with adjacent tRNA (MTT) genes (mt.15956-647), the human ND1 gene with an in frame GFP gene and adjacent endogenous MTT genes and heterologous rat MTT genes. Natural competence of isolated human mitochondria of HepG2 cells was used to import reporter constructs. The import efficiency of various fluorescently labelled PCR-generated import substrates in the range of 250bp up to 3.5kb was assessed by quantitative PCR and evaluated by confocal microscopy. Heterologous expression of the imported construct was confirmed at RNA level by a circular RNA (cRNA)-RT-PCR assay for the expression of tRNAs and by in organello [α-(32)P]-UTP labelling and subsequent hybridisation to reporter-specific sequences for monitoring mRNA expression. Heterologous expression of rat mitochondrial tRNA(Leu(UUR)) (rMT-TL1) was confirmed by co-/post-transcriptional trinucleotide (CCA) addition. Interestingly, the rat-specific MT-TL1 was correctly processed in isolated human mitochondria at the 3' end, but showed an aberrant 5' end processing. Correct 3' end processing of the heterologous expressed mitochondrial rat tRNA(Ser2) (MT-TS2) was detected. These findings demonstrate the feasibility of genetic manipulation of human mitochondria, providing a tool for characterisation of cis-acting elements of the human mitochondrial genome and for the study of human mitochondrial tRNA processing in organello.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Unit Childrens Hospital > Forschungsgruppe Humangenetik
04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Paediatric Medicine
04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Institute of Clinical Chemistry

UniBE Contributor:

Jackson, Christopher; Gallati, Sabina and Schaller, André

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1567-7249

Publisher:

Elsevier

Language:

English

Submitter:

André Schaller

Date Deposited:

19 Mar 2015 12:32

Last Modified:

29 Oct 2015 10:17

Publisher DOI:

10.1016/j.mito.2014.05.011

PubMed ID:

24911383

Uncontrolled Keywords:

Heterologous expression, In organello, Mitochondria, Mitochondrial gene expression, Mitochondrial transfection, Natural competence

BORIS DOI:

10.7892/boris.65358

URI:

https://boris.unibe.ch/id/eprint/65358

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